Antibodies that specifically react with seroreactive regions on HPV 16 proteins E1 and E2

ABSTRACT

The invention relates to seroreactive regions on the E1 and E2 proteins of human papillomavirus 16 (HPV 16). The invention also relates to a vaccine that contains peptides comprising at least one seroreactive region of the E1 and/or E2 proteins. The invention likewise embraces compositions for diagnostic purposes, which contain peptides with the seroreactive regions. Further, the invention relates to antibodies that bind to seroreactive regions of the E1 and/or E2 proteins of HPV 16, and compositions comprising such antibodies, which can be used for diagnostic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.08/468,337, filed Jun. 6, 1995, now U.S. Pat. No. 6,221,577, which is adivisional of U.S. application Ser. No. 08/237,418, filed May 3, 1994,now U.S. Pat. No. 5,601,973, which is a continuation of application Ser.No. 07/913,613, filed Jul. 16, 1992, now abandoned. Applicants rely onthe disclosures, and claim the benefit of the filing date, of all ofthese applications.

The invention relates to seroreactive regions on proteins E1 and E2 ofhuman papillomavirus (HPV) 16.

The application also relates to a vaccine which contains such peptideswhich contain the seroreactive regions.

The invention likewise embraces compositions for diagnostic purposeswhich contain peptides with the seroreactive regions.

HPV 16 is one of the human papillomaviruses (Proc. Natl. Acad. Sci., USA80, 3813-3815 (1983). The organization of the genome of HPV 16 has beendescribed in Virology 145, 181-185 (1985).

Genomic sequences of HPV can be detected in most cases of preinvasiveand invasive cervical tumors. HPV 16 has been identified world-wide asthe virus type predominating in these tumors. The HPV 16 genome isdetectable in more than 50% of cervical tumors, in which case it isoften present integrated into the cellular DNA. Little is known aboutthe immune response after infections with HPV 16 or otherpapillomaviruses.

DESCRIPTION OF THE INVENTION

Initial data: patients suffering from cervical tumors were compared withhealthy individuals with regard to the presence of antibodies againstviral proteins. These viral proteins were then linked as fusion productswith various prokaryotic peptides at their N terminus and then used asantigens in Western blots.

The object of the present invention is the further identification of HPV16 viral structures which can be used as tool in the prophylaxis,diagnosis and therapy of HPV 16-dependent tumorous diseases in humans.The identification of such structures is a prerequisite for thedevelopment of ELISAs which make it possible to test a large quantity ofhuman sera for the presence of HPV 16.

The present invention therefore embraces seroreactive regions of the E1protein of HPV 16, which have one of the following amino-acid sequences:

I. NGWFYVEAVVEKKTGDAISDDENENDSDTGEDVDFIVNDNDYLT (SEQ ID NO:1)

II. NENDSDTGEDLVDFIVND (SEQ ID NO:2)

III. MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDYLT (SEQ IDNO:3)

IV. EDLVDFIVNDNDYLT (SEQ ID NO:4)

V. EDLVDFIVNDNDYLTQAETETAHALFTAQEAKQH (SEQ ID NO:5)

VI. NENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLKRKYL (SEQ ID NO:6)

VII. GSPLSDIS (SEQ ID NO:7);

seroreactive regions of the E2 protein of HPV 16, which have one of thefollowing amino-acid sequences:

I. DKILTHYENDS (SEQ ID NO:8)

II. DKILTHYRENDSTDLRDHI (SEQ ID NO:9)

III. DLRDHIDYWKH (SEQ ID NO:10)

IV. AIYYKAREMGFKHINHQVVPTLA (SEQ ID NO:11)

V. AIYYKAREMGFKHINHQVVPTLAVSKNKAL (SEQ ID NO:12)

VI. YYKAREMGFKHINHQVVPTLAVSKN (SEQ ID NO:13)

VII. INHQVVPTLAVSKNKAL (SEQ ID NO:14)

VIII. INHQVVPTLAVSKNKAL (SEQ ID NO:15)

IX. TLAVSKNKALQAIELQLTLETIYNSQYSNEKWTQDV (SEQ ID NO:16)

X. QLTLETIYNSQOYSNKWTLQDVSLE (SEQ ID NO:17)

XI. TLETIYNSQYSNEK (SEQ ID NO:18)

XII. TSVFSSNEVSSPEII (SEQ ID NO:19)

XIII. VFSSNEVSSPEIIRQHLANHP AATHTKAVALGTEET (SEQ ID NO:20)

XIV. EIIRQHLNHPAATHTKAVALGTEETQTTIOPRSEP (SEQ ID NO:21)

XV. TEETQTTIQRPRSEPDTGN (SEQ ID NO:22)

The invention furthermore embraces peptides with one or more of theseroreactive regions identified above, a vaccine which contains one ormore of the peptides identified above, a composition for diagnosticpurposes for the identification of specific antibodies against HPV E1and/or E2 protein, which likewise contain the peptides identified above,and monoclonal antibodies which have an affinity for one or more of theseroreactive regions of the E1 or E2 protein of HPV 16, and acomposition for diagnostic purposes which contains these monoclonalantibodies.

In order to identify seroreactive regions in proteins El and E2 of HPV,the experimental route described in Science 228, 1315-1317 (1985) wasfollowed. Subgenomic HPV 16 DNA fragments which had been randomlygenerated by ultrasound treatment and partial DNAse I treatment werecloned into the phage vector fuse1 and then expressed as part of a phagecoat protein. Seroreactive phage recombinants were identified using seraprepared against E1 and E2, and purified, and the seroreactive regionswere characterized by sequencing the HPV 16 portion. Polyclonal rabbitsera against an HPV 16 E1 MS2 polymerase fusion protein and against theamino- and carboxyl-terminal part of HPV 16 E2 (separate, likewise MS2fusion proteins) were prepared.

The filamentous phages embrace the three groups fl, fd and M13. It iscommon to them all that binding and uptake of the phages takes place viaF pili of the bacteria, i.e. that only F⁺ strains can be infected. Thefd wild-type phage, from which the vector system used is derived, formsparticles which are about 900×6 nm in size and which are composed inparticular of about 2700 subunits of the main coat protein. In addition,in each case 5 molecules of the minor coat proteins pIII, pVI, pVII andpIX are located at both ends of the virions. The single-stranded,circular phage genome which, in the case of the fd wild-type, is 6408 bpin size, carries the information for a total of 10 different proteins.

In the fd derivatives fuse1, fuse2 (Parmley and Smith, Gene, 7, 305-318(1988)) and fusemm, a tetracycline-resistance gene is integrated, byinsertion of a part of the Tn10 transposon, in the phage genome, whichhas been enlarged to about 9.2 kbp in this way. This means that thereplicative DNA double-stranded phage genomes behave in the bacterialike selectable plasmids and can accordingly be prepared and used forclonings. Another modification from the wild-type is the presence of areading frame mutation in the gene for the minor coat protein pIII inconjunction with an inserted restriction site for cloning expressableDNA fragments. The gene for pIII is composed of two almost completelyindependent domains (Crissmann and Smith, 1984): an N-terminal domainwhich mediates the binding of the phages to the bacterial cell receptor(F pili) and a C-terminal protein domain which is responsible for phagemorphogenesis. The reading frame mutation, which is located directlybehind the signal sequence of the protein, thus leads to inactivation ofthe gene and accordingly also prevents the formation of infectiousparticles. This is of importancefor the replication of these phagemutants as plasmids because the fd genomes inactivated in themorphogenesis do not damage the host bacteria (Smith, in: Vectors, ASurvey of Molecular Cloning Vectors and Their Uses, ButterworthPublishers, Stoneham, Mass. 61-85, 1987).

Insertion of suitable DNA fragments and restoration of gene IIIfunctions lead to the formation of infectious phage particles whichcarry additional amino-acid sequences on their coats. These sequencesare accessible to various ligands, for example antibodies, in thenatural state of the phages.

The fd expression system used in this invention is essentially based onsetting up phage banks by cloning DNA foreign sequences into the geneIII, and examining the latter with the aid of monoclonal or polyclonalsera for seroreactive recombinants. An amplification normally takesplace on preparation of these expression banks. The extent of thisreplication of individual clones in turn depends on the nature and sizeof the inserted DNA sequence. This means that different clones differ infrequency, which may differ by up to several powers of ten. It istherefore possible to derive from the stated properties the followingtwo features of the fd expression banks:

Amplification of the banks, which leads to repeated cloning of identicalphage clones isolated by immunoscreening.

Possibility of enriching seroreactive phages by affinity chromatography(columns) because phages in the active state can be bound and elutedagain.

The repeated isolation of identical recombinants was avoided by usingseparately set up banks, there being an extremely low probability ofcloning a DNA fragment prepared identically and in parallel, or of thephage recombinant derived therefrom.

In this invention, a total of 11 different expression banks for HPV 16DNA in fuse1 were set up. The number of primary, tetracycline-resistantand insert-harbouring recombinants was in this case between 2000 and90000 per bank. Since complete plasmids composed of about 4 kb vectorportion and 8 kb HPV portion in sheared form were always used for thecloning, the HPV-containing fd recombinants are reduced by about 30%.The fragments cloned in were then expressed, as already mentioned, asfusion protein of the gene III coat protein. The cloning site in thegene III is in this case directly behind the translated signal sequencefor protein export. In order to restore the function of the gene it isnecessary for an insert to have a defined size (3n+2; n=0, 1, 2, 3 . . .). In order to express a defined protein sequence as fusion protein ofthe gene III product it is necessary in addition for both the 3′ and the5′ junction to be in the correct reading frame, and for thecorresponding insert to be present in the correct orientation. Thistherefore applies overall to only about every 18th (3×3×2) HPVDNA-containing recombinants. A small portion thereof is in turninactivated by translation stop codons present in the insert or byproteins which are not functional because of their folding. Because ofthe stated parameters it is difficult to estimate what is the minimumnumber of recombinants necessary to express with great probability anyrequired part of an HPV genome as fd fusion protein in the phage bank.In papillomaviruses about 10 kb of the genome (partly by overlappingopen reading frames) code for proteins. Of 2000 tetracyclineresistantinsert-harboring recombinants, about 100 (1/18) clones express HPVsequences in a suitable manner. With average HPV fragment sizes of50-150 bp, the expressed HPV sequence amounts to about 5000-15000 bp. Infact fd banks with about 2000 recombinants prove to be sufficientlyrepresentative.

In order to ensure the specificity of the immunoscreening, eitherseveral different recombinants of a seroreactive region or at leastseveral identical but independently isolated phage recombinants werealways isolated.

The amino-acid position indicators in FIGS. 2 and 5 hereinafter relateto the E1 and E2 proteins and not to the positions of the open readingframes. The first methionine was given position 1.

EXAMPLE 1 Preparation of Polyclonal Antisera Against HPV 16 E1

In order to isolate seroreactive phage recombinants from the HPV 16 fdexpression bank, initially polyclonal rabbit sera against HPV 16 E1 MS2fusion proteins were prepared. For this, the Pst I A fragment of HPV 16(bp 875-3693) was cloned into the Pst I cleavage site of the expressionvector pEX12mer (Seedorf et al., EMBO J. 6, 139-144, 1987), by whichamino acids 5-649 of HPV 16 E1 ORF are expressed (FIG. 1). This vectoris a derivative of the plasmid pPLC24 (Remaut et al., Gene 15, 81-93,1981) which has been modified by insertion of the pUC8 polylinker behindthe MS2 polymerase portion. The fusion protein is transcribed in thepEX12mer by the temperature-inducible lambda pL promoter. The N-terminalfusion portion of the MS2 protein amounts to 100 amino acids.

Since the original HPV 16 isolate (Seedorf et al., Virology, 145,181-185, 1985) has a reading frame mutation in the region of the E1 openreading frame (nucleotide position 1138), recourse was had to an HPV 16isolate from a cervical carcinoma with a complete E1 ORF. Because of theselected restriction cleavages, the HPV 16 E1 open reading frame (bp865-2811) is thus completely expressed apart from three N-terminal aminoacids.

The clonings and plasmid analyses were initially carried out using theE. coli strain W6 in which there is constitutive expression of therepressor for the lambda promoter. This prevented expression of thefusion proteins, in order to prevent counterselection after thetransformations. After examination of the cloning by restrictionanalysis, and Southern blot hybridization with radioactive labelled HPV16 DNA (Pst I A fragment), the plasmid DNA of the construct was used fortransformation in E. coli N6045. This strain is able, because of itstemperature-sensitive repressor of the lambda promoter, to express theMS2 fusion proteins.

It was then possible in a Western blot to examine, with the aid of amonoclonal antibody directed against the MS2 portion of the fusionprotein, by comparison of extracts from induced and non-induced bacteriathe size and the expression rate of the fusion protein. Since the bandof the MS2 E1 fusion protein corresponded to the expected size of about90 kD, no examination of the cloning junctions by sequencing was carriedout. In the two other reading frames of the HPV 16 Pst I A fragment,expression of larger proteins is impossible because of the presence oftranslation stop codons. In addition, both Pst I cleavage sites of thevector-insert junctions were retained. Correct expression of the E1 openreading frame was confirmed by the results of the immunoscreening of theHPV 16 fd expression banks, which are described in the followingsection.

The MS2-E1 fusion protein was then purified from induced E. colicultures by differential extraction and by electroelution from SDSpolyacrylamide gels, and was then used to immunize two rabbits.

EXAMPLE 2 Identification of Seroreactive Regions on the HPV 16 E1Protein

Both of the polyclonal rabbit sera prepared against HPV 16 E1 were usedto examine five different HPV 16 fd expression banks for reactiverecombinants. It was possible in this way to identify a total of atleast two different antibody binding sites represented bynon-overlapping phage clones. In total, 19 independent phage cloneswhich contain seven different classes of HPV 16 inserts were isolated(FIG. 2). Six classes have a common overlapping region which codes forthe HPV 16 E1 specific peptide EDLVDFIVND (SEQ ID NO:23). The secondidentified epitope on the E1 protein is represented by a recombinantphage (clone 1059) which codes for the E1 peptide GSPLSDIS (SEQ IDNO:7).

The original HPV 16 isolate has a reading frame mutation in the E1 openreading frame (nucleotide position 1138). The DNA of this HPV 16 isolatewas used to prepare the fd expression banks. Two of the isolatedseroreactive fd recombinants contain this region and therefore also havethe reading frame mutation. In clone 1145 this leads to a change ofreading frame, and this results in C-terminal attachment of three HPV16-E2 non-specific amino acids ( . . . ValValHis). Clone 1059 starts inthe wrong frame and is converted into the correct HPV 16 E1 readingframe by the reading frame mutation of the HPV 16 isolate used. Theclone codes for the peptide STGKTKVFGSPLSDIS (SEQ ID NO:24) of whichonly the C-terminal amino acids . . . GSPLSDIS (SEQ ID NO:7) derive fromthe actual HPV 16 E1 protein and must form the epitope.

Both clones which contain the reading frame mutation have the correctinsert size (3n+2 base pairs) to restore the reading frame of gene IIIof the phage vector.

EXAMPLE 3 Preparation of Polyclonal Antisera Against HPV 16 E2

Like the case of the HPV 16 E1 open reading frame, no suitable antiserawere available for the HPV 16 E2 protein either. For this reason, theHPV 16 E2 open reading frame (nucleotide position 2756-3850; AA 1-365)was expressed in the-vector pEX12mer as already described for the E1protein.

Firstly the HPV 16 DNA fragment was cloned via the Hinf I cleavage siteat position 2761 into the pEX12mer vector. In this case the startingmaterial was an already subcloned HPV 16 fragment (bp 2367-4467). Thisfragment was cut out of the vector again, via the additionally insertednon-HPV 16-specific restriction sites Xba I (5′ end) and Bam HI (3′end), and prepared. This DNA fragment which is 2.1 kb in size (Xba I/BamHI) was then partially cut with Hinf I. This results, inter alia, in afragment which is 1700 bp in size between the 3′-terminal Ban HIcleavage site and Hinf I site at bp 2761. The internal Hinf I cleavagesite (bp 3539) in this fragment is uncleaved, and the HPV 16 E2 ORF iscompletely present apart from three amino-terminal amino acids. Afterpreparation, the Hinf/Bam fragment was cloned into the pEX12merexpression vector which had been cleaved with Bam HI. This resulted, viathe compatible Bam HI sites, in linear products of vector and insert.The free ends of these products were filled in with Klenow polymeraseand then closed by ligation. This results in an MS2-E2 junction at thefilled-in cleavage sites Bam HI (vector) and Hinf I (E2 insert) withloss of the two restriction sites. Using Eco RI/Bam HI doublerestriction cleavages it was possible to identify recombinants whichharboured the HPV 16 E2 fragment in the correct orientation.

After transformation into the E. coli expression strain 6045 it was notpossible using a monoclonal antibody directed against the MS2 polymeraseto find any production whatever of the MS2 fusion protein. In order torule out a displacement of the reading frame at the NS2-E2 junction, theplasmid DNA of a total of 16 different MS2-E2 recombinants washybridized in a Southern blot with an oligonucleotide derived from thecorrect Ban HI/Hinf I junction. Since an unambiguous hybridizationsignal was identifiable with 15 clones, it was assumed that the cloninghad taken place in the correct reading frame, and expression of thecomplete E2 ORF is not possible in pEX vectors. As a substitute, the HPV16 E2 protein was then expressed in two halves in the pEX12mer vector.

EXAMPLE 4 Expression of the Amino-terminal Region of HPV 16 E2

The amino-terminal region of the E2 open reading frame betweennucleotide position 2761 and 3209 was cloned into the pex12mer vectorand expressed. Since the E2 open reading frame starts at nucleotideposition 2756, the MS2-E2 fusion protein lacks the first two amino acids(Met-Glu) of the E2 protein (FIG. 4).

Plasmid DNA composed of pEX12mer and HPV 16 E2, which were obtained fromthe cloning described above, was truncated at the carboxyl terminus bydeletion of a Hinc II (HPV 16 bp 3209)/Bam HI fragment and religation(blunt/flush from Hinc II and Bam HI). This results in expression of theN-terminal part of HPV 16 E2 between nucleotide position 2761 (Hinf I)and 3209 (Hinc II). A fusion protein about 30 kD in size was detectablein induced bacteria in a Western blot with an anti-MS2 moleculeantibody.

The fusion protein was purified by differential extraction of theinduced bacterial lysate and by electroelution of the protein band fromSDS polyacrylamide gels stained with Coomassie blue, and used forimmunizing rabbits.

EXAMPLE 5 Expression of the Carboxyl-terminal Region of HPV 16 E2

The C-terminal region of the HPV 16 E2 open reading frame betweennucleotide position 3209 and 3850 was expressed in the pEX12mer vector(FIG. 3). The region is thus directly connected to the expressedamino-terminal part, described above, of the HPV16 E2 open readingframe.

For this, recourse was had to the Xba/Bam fragment which has beendescribed above and which contains the complete HPV 16 E2 reading frame.After restriction cleavage, a Hinc II/Bam HI fragment (nucleotideposition 3209-4467) which contains the carboxyl-terminal half of HPV 16E2 was isolated. This fragment was inserted into the Bam HI cleavagesite of the pEX12mer expression vector (5′ Bam HI/Hinc II—Bam HI/Bam HI3′). It was possible with the aid of the anti-MS2 monoclonal antibody toidentify in extracts of induced bacteria a fusion protein of about 30kD, which was purified by differential extraction and electroelutionfrom SDS polyacrylamide gels, and was used to immunize rabbits.

EXAMPLE 6 Identification of Seroreactive Regions on the HPV 16 E2Protein

Available for the immunoscreening of the fd HPV 16 expression banks wasa total of four different anti-HPV 16 E2 antisera: in each case two seraagainst the amino-terminal part (bp 2761-3209; AA 3-152) and two againstthe carboxyl-terminal part of the E2 (bp 3209-3850; AA 153-365) openreading frame. These sera were used to examine five different expressionbanks for seroreactive recombinants. This resulted in isolation of atotal of 32 clones, of which 26 contain amino-terminal sequences of theE2 protein. These 26 clones form a total of 11 different classes whichrepresent four different non-overlapping regions (FIG. 5).

All the epitopes are located in a restricted region comprising 88 aminoacids of the amino terminus of the E2 open reading frame which islocated between nucleotide position 2792 (AspLysIle . . . ) and 3055 ( .. . SerLeuGlu).

It was possible to locate in the carboxyl-terminal region at least twoindependent non-overlapping epitopes (TSVFSSNEVSSPEII (SEQ ID NO:19) andTEETQTTIQRPRISEPDTGM, (SEQ ID NO:20) FIG. 5). These are represented by atotal of four classes of recombinants with six independent isolates. Theregion of the E2 open reading frame which is covered by the clones islocated between nucleotide position 3343 (ThrSerVal . . . ) and 3502 ( .. . ThrGlyAsn) and comprises 52 amino acids.

Five classes of recombinants (12 isolates) extend over nucleotideposition 2926. All the clones have a point mutation (A→G transition)here, but this does not lead to a change in the corresponding amino acid(glutamine).

EXAMPLE 7 Immunoscreening of Fd Phase Expression Banks

1. Phage Affinity Concentration with Protein A-Sepharose Columns

The phage banks prepared in the fd phage expression system usedunavoidably underwent amplification on cloning. The extent of thisreplication of the original clones is in turn greatly influenced by thenature of the individual recombinants, for example by different sizes ofinserts or conformation of the coat proteins, inhibition ofphysiological processes in the infected bacteria and many others, and itwas therefore not to be expected that uniform amplification of allphages takes place. In order to isolate underrepresented phagerecombinants or clones from large libraries, seroreactive phagerecombinants were concentrated. For this, use was made of thecircumstance that the foreign sequences expressed in each case appear aspart of an fd gene III fusion protein on the coat of natural phageparticles. Large amounts of phages (10⁹-10¹² particles) were for thispurpose bound to protein A antibody columns and eluted again.

For this, initially protein A-Sepharose was swollen with PBS for 30 minand was washed with PBS. Subsequently the protein A-Sepharose wasincubated with about 1 to 2 ml of suitable polyclonal sera (rabbit orhuman) or with corresponding protein A-binding monoclonal antibodies inEppendorf reaction tubes on a rotary shaker at 4° C. for 1 to 2 days.Subsequently the protein A-Sepharose was washed 10 times by theSepharose being alternately resuspended in 10 ml of PBS and pelletedagain by centrifugation (2 min, 6000 rpm). The protein A-Sepharose-IgGcomplexes formed were then incubated with an appropriate amount ofphages as above. Then the Sepharose.was washed with PBS several times asbefore and packed into a Pasteur pipette closed with a glass bead andwashed with several liters (2-15 l) flowing through. The column materialwas removed and then incubated in the same volume of elution buffer (1mg/ml BSA, 0.1 M HCl, glycine, pH 2.2) for 15 min. After briefcentrifugation the supernatant, which now contains free phages andantibodies, was neutralized with 1/5 of the volume of tris base (0.5 M).Antibodies which recognize the recombinant gene are able to inhibitbinding of the phage to the bacterial cells and thus the cycle ofinfection. For this reason the phages were added in 100-200 μl aliquotsof the eluates immediately after neutralization to exponentially growingE. coli K91 and plated out on complete medium plates. It emerged duringthe work that replica filters of these phage platings were unsuitablefor immunoblotting, probably because of contaminants in the eluate. Forthis reason the resulting plaques were again rinsed off the plates withcomplete medium and subsequently plated out from the phage suspensionsobtained in this way, which had undergone renewed amplification, and theimmunoblotting was carried out on minimal agar plates.

2. Phage Platings and Preparation of Nitrocellulose Replica Filters forthe Immunoblotting

All the fd phage derivatives were plated out on a lawn formed by E. coliK91 (Lyons and Zinder, Virology, 49, 45-60, 1972). This strain isdistinguished by a large number of F pili (5 per cell, compared withabout 0.5 per cell in most F⁺ strains) which are responsible for uptakeof filamentous phages. This is particularly important for the fdexpression system used in this study because the recombinant fuse phageshave, owing to the uptake of a part of the Tn10 transposon (tetracyclineresistance), a genome which is distinctly enlarged compared with thewild-type, and for this reason form particularly small plaques.

To plate out the phages, a K91 overnight culture was diluted 1:100 incomplete medium (2× YT) and incubated at 37° C. for 3 to 4 h. After adensity of E₆₀₀=0.8-1.2 was reached, 200 μl of the bacteria were platedout with an appropriate amount of phages, together with 3.5 ml ofagarose (0.6% agarose, 10 mM XgSO₄, 50° C.) on prewarmed bacteriaplates. Minimal agar plates were always used for every plating intendedto be used for nitrocellulose replicas for the immunoscreening. Platingsout for determination of the phage titer or for DNA hybridization werecarried out on complete medium plates.

Use of complete medium plates for the immunoblotting always lead to veryhigh non-specific reactivity of the filters with the sera used.

The plates were incubated at 37° C. overnight. After about 16 hours, anitrocellulose filter was placed on for 10-15 min, marked with fourasymmetric pricks with a needle and removed again using flat-endedforceps. The filters were labelled and then inverted onto a freshminimal agar plate and incubated further at 37° C. for 5-6 hours. Thisincreased the amount of phage particles (proteins) on the filter sincethe reincubation makes it possible for the bacteria and phages bound tothe filters to grow further via the nutrients diffusing from the plate.Subsequently the filters were removed and saturated in 10% milk (skimmilk powder in PBS) for 30-60 min. The filters were then incubated withsuitable dilutions of appropriate sera in 5% milk at 4° C. overnight.

3. Immunostaining of Replica Filters and Cloning of ReactiveRecombinants

After removal of the replica filters, blocking in 10% milk (in PBS) for60 min and overnight incubation with antisera, the nitrocellulosefilters were washed in PBS, 0.05% Tween 20 (5 changes of washing buffer)for 30 min. The filters were then incubated with 1:1000 dilutions ofappropriate second antibodies (peroxidase-coupled goat anti-human,anti-rabbit or anti-mouse) in 5% milk at RT for 2 h. This was followedby renewed washing (see above) and incubation in the following stainingmixture:

40 mg of diaminobenzidine 30 μl of 30% of H₂O₂ 1.5 ml of 1% NiSO₄ in 50ml of PBS

After sufficient color had developed, the filters were removed from thesolution, placed in water for 30 min and then dried on 3MM paper.

The prick holes and signals on the filters were then copied onto a sheetor the lid of a bacteria dish. This made it possible to assign aposition or, if the phage dilution was sufficiently large (round 2 orhigher), a plaque to a signal. A sterile toothpick was gently stabbedinto the position or the plaque, and the toothpick was placed in 500 μlof complete medium for 10-15 min. This phage suspension then containedgenerally about 10⁶-10⁷ infectious particles, which comprises about0.1-1% of the phages in a plaque. The phage suspensions were thenincubated at 65° C. for 15-20 min in order to kill bacteria which hadbeen carried over, and were then stored at 4° C.

DESCRIPTION OF THE FIGURES

FIG. 1

Cloning of the E1 open reading frame into the expression vectorpEX12mer. The figure includes DNA sequences SEQ ID NO:25, SEQ ID NO:27,SEQ ID NO:29, SEQ ID NO:31, and amino acid sequences SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, and SEQ ID NO:32.;

FIG. 2

Seroreactive regions on the HPV 16 E1 protein. The figure includes aminoacid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, and SEQ ID NO:24.; Small letters indicate theamino acids of clones 1145 and 1059 which, because of the change inreading frame of the HPV 16 isolate used for cloning the fd banks, arenot derived from the HPV 16 E1 protein (see text). Clones 1090, 1079,1084, 1029, 1099 and 1145 have a common region of 10 amino acids(EDLVDFIVND) (SEQ ID NO:23) which possibly represents a common epitopeof the clones, although other antibody binding sites on these clonescannot be ruled out. Clone 1059 has, because of the change in readingframe, no common amino-acid sequences with the other clones, althoughthe insert of this clone overlaps with the insert of clone 1145. Theposition indications relate to the HPV 16 E1 open reading frame. Theamino acids of clones 1145 and 1059 which do not derive from E1 are nottaken into account here.

FIG. 3

Cloning of the carboxyl-terminal half of the HPV 16 E2 protein into theexpression vector pEX12mer. ID NO:25, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, and amino acid sequences SEQ ID NO:26, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40.;

FIG. 4

Cloning of the amino-terminal half of the HPV 16 E2 protein into theexpression vector pEX12mer. The figure includes DNA sequences SEQ IDNO:25, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:43 and aminoacid sequences SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:42,and SEQ ID NO:44.;

FIG. 5

Seroreactive regions on the HPV 16 E2 protein., The figure includesamino acid sequences SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, and SEQ ID NO:22.

The regions (E2-1066, -1170, -1074, -1112) on the carboxyl-terminal halfof HPV 16 E2 are all located in a region 88 amino-acids long (between AA13 and 100) and partially overlap. The carboxyl-terminal regions arealso closely adjacent (between AA 197 and 249). The two regions are ineach case arranged approximately proportional to their position on theE2 protein.

44 45 amino acids amino acid linear peptide 1 Asn Gly Trp Phe Tyr ValGlu Ala Val Val Glu Lys Lys Thr Gly Asp 1 5 10 15 Ala Ile Ser Asp AspGlu Asn Glu Asn Asp Ser Asp Thr Gly Glu Asp 20 25 30 Leu Val Asp Phe IleVal Asn Asp Asn Asp Tyr Leu Thr 35 40 45 18 amino acids amino acidlinear peptide 2 Asn Glu Asn Asp Ser Asp Thr Gly Glu Asp Leu Val Asp PheIle Val 1 5 10 15 Asn Asp 60 amino acids amino acid linear peptide 3 MetAla Asp Pro Ala Gly Thr Asn Gly Glu Glu Gly Thr Gly Cys Asn 1 5 10 15Gly Trp Phe Tyr Val Glu Ala Val Val Glu Lys Lys Thr Gly Asp Ala 20 25 30Ile Ser Asp Asp Glu Asn Glu Asn Asp Ser Asp Thr Gly Glu Asp Leu 35 40 45Val Asp Phe Ile Val Asn Asp Asn Asp Tyr Leu Thr 50 55 60 15 amino acidsamino acid linear peptide 4 Glu Asp Leu Val Asp Phe Ile Val Asn Asp AsnAsp Tyr Leu Thr 1 5 10 15 34 amino acids amino acid linear peptide 5 GluAsp Leu Val Asp Phe Ile Val Asn Asp Asn Asp Tyr Leu Thr Gln 1 5 10 15Ala Glu Thr Glu Thr Ala His Ala Leu Phe Thr Ala Gln Glu Ala Lys 20 25 30Gln His 54 amino acids amino acid linear peptide 6 Asn Glu Asn Asp SerAsp Thr Gly Glu Asp Leu Val Asp Phe Ile Val 1 5 10 15 Asn Asp Asn AspTyr Leu Thr Gln Ala Glu Thr Glu Thr Ala His Ala 20 25 30 Leu Phe Thr AlaGln Glu Ala Lys Gln His Arg Asp Ala Val Gln Val 35 40 45 Leu Lys Arg LysTyr Leu 50 8 amino acids amino acid linear peptide 7 Gly Ser Pro Leu SerAsp Ile Ser 1 5 11 amino acids amino acid linear peptide 8 Asp Lys IleLeu Thr His Tyr Glu Asn Asp Ser 1 5 10 18 amino acids amino acid linearpeptide 9 Asp Lys Ile Leu Thr His Tyr Glu Asn Asp Ser Thr Asp Leu ArgAsp 1 5 10 15 His Ile 11 amino acids amino acid linear peptide 10 AspLeu Arg Asp His Ile Asp Tyr Trp Lys His 1 5 10 23 amino acids amino acidlinear peptide 11 Ala Ile Tyr Tyr Lys Ala Arg Glu Met Gly Phe Lys HisIle Asn His 1 5 10 15 Gln Val Val Pro Thr Leu Ala 20 30 amino acidsamino acid linear peptide 12 Ala Ile Tyr Tyr Lys Ala Arg Glu Met Gly PheLys His Ile Asn His 1 5 10 15 Gln Val Val Pro Thr Leu Ala Val Ser LysAsn Lys Ala Leu 20 25 30 25 amino acids amino acid linear peptide 13 TyrTyr Lys Ala Arg Glu Met Gly Phe Lys His Ile Asn His Gln Val 1 5 10 15Val Pro Thr Leu Ala Val Ser Lys Asn 20 25 20 amino acids amino acidlinear peptide 14 Ile Asn His Gln Val Val Pro Thr Leu Ala Val Ser LysAsn Lys Ala 1 5 10 15 Leu Gln Ala Ile 20 17 amino acids amino acidlinear peptide 15 Ile Asn His Gln Val Val Pro Thr Leu Ala Val Ser LysAsn Lys Ala 1 5 10 15 Leu 37 amino acids amino acid linear peptide 16Thr Leu Ala Val Ser Lys Asn Lys Ala Leu Gln Ala Ile Glu Leu Gln 1 5 1015 Leu Thr Leu Glu Thr Ile Tyr Asn Ser Gln Tyr Ser Asn Glu Lys Trp 20 2530 Thr Leu Gln Asp Val 35 25 amino acids amino acid linear peptide 17Gln Leu Thr Leu Glu Thr Ile Tyr Asn Ser Gln Tyr Ser Asn Glu Lys 1 5 1015 Trp Thr Leu Gln Asp Val Ser Leu Glu 20 25 14 amino acids amino acidlinear peptide 18 Thr Leu Glu Thr Ile Tyr Asn Ser Gln Tyr Ser Asn GluLys 1 5 10 15 amino acids amino acid linear peptide 19 Thr Ser Val PheSer Ser Asn Glu Val Ser Ser Pro Glu Ile Ile 1 5 10 15 36 amino acidsamino acid linear peptide 20 Val Phe Ser Ser Asn Glu Val Ser Ser Pro GluIle Ile Arg Gln His 1 5 10 15 Leu Ala Asn His Pro Ala Ala Thr His ThrLys Ala Val Ala Leu Gly 20 25 30 Thr Glu Glu Thr 35 37 amino acids aminoacid linear peptide 21 Glu Ile Ile Arg Gln His Leu Ala Asn His Pro AlaAla Thr His Thr 1 5 10 15 Lys Ala Val Ala Leu Gly Thr Glu Glu Thr GlnThr Thr Ile Gln Arg 20 25 30 Pro Arg Ser Glu Pro 35 19 amino acids aminoacid linear peptide 22 Thr Glu Glu Thr Gln Thr Thr Ile Gln Arg Pro ArgSer Glu Pro Asp 1 5 10 15 Thr Gly Asn 10 amino acids amino acid linearpeptide 23 Glu Asp Leu Val Asp Phe Ile Val Asn Asp 1 5 10 17 amino acidsamino acid linear peptide 24 Ser Thr Gly Ser Lys Thr Lys Val Phe Gly SerPro Leu Ser Asp Ile 1 5 10 15 Ser 51 base pairs nucleic acid doublelinear DNA (genomic) 25 TTGTCATGGG ATCTGAATTC CGGGGGGATC CGTCGACCTGCAGCCAAGCT T 51 17 amino acids amino acid linear peptide 26 Leu Ser TrpAsp Leu Asn Ser Gly Gly Ile Arg Arg Pro Ala Ala Lys 1 5 10 15 Leu 42base pairs nucleic acid double linear DNA (genomic) 27 TTGTCATGGGATCTGAATTC CGGGGGGATC CGTCGACCTG CA 42 14 amino acids amino acid linearpeptide 28 Leu Ser Trp Asp Leu Asn Ser Gly Gly Ile Arg Arg Pro Ala 1 510 33 base pairs nucleic acid double linear DNA (genomic) 29 GGTACCAATGGGGAAGAGGG TACGGGATGT AAT 33 12 amino acids amino acid linear peptide 30Ala Gly Thr Asn Gly Glu Glu Gly Thr Gly Cys Asn 1 5 10 75 base pairsnucleic acid double linear DNA (genomic) 31 TTGTCATGGG ATCTGAATTCCGGGGGGATC CGTCGACCTG CAGGTACCAA TGGGGAAGAG 60 GGTACGGGAT GTAAT 75 25amino acids amino acid linear peptide 32 Leu Ser Trp Asp Leu Asn Ser GlyGly Ile Arg Arg Pro Ala Gly Thr 1 5 10 15 Asn Gly Glu Glu Gly Thr GlyCys Asn 20 25 26 base pairs nucleic acid double linear DNA (genomic) 33TTGTCATGGG ATCTGAATTC CGGGGG 26 10 amino acids amino acid linear peptide34 Leu Ser Trp Asp Leu Asn Ser Gly Gly Ile 1 5 10 30 base pairs nucleicacid double linear DNA (genomic) 35 TTGTCATGGG ATCTGAATTC CGGGGGGATC 3010 amino acids amino acid linear peptide 36 Leu Ser Trp Asp Leu Asn SerGly Gly Ile 1 5 10 27 base pairs nucleic acid double linear DNA(genomic) 37 ACTCTTTGCC AACGTTTAAA TGTGTGT 27 9 amino acids amino acidlinear peptide 38 Thr Leu Cys Gln Arg Leu Asn Val Cys 1 5 57 base pairsnucleic acid double linear DNA (genomic) 39 TTGTCATGGG ATCTGAATTCCGGGGGGATC ACTCTTTGCC AACGTTTAAA TGTGTGT 57 19 amino acids amino acidlinear peptide 40 Leu Ser Trp Asp Leu Asn Ser Gly Gly Ile Thr Leu CysGln Arg Leu 1 5 10 15 Asn Val Cys 30 base pairs nucleic acid doublelinear DNA (genomic) 41 GACTATTATG GTTTATATTA TGTTCATGAA 30 10 aminoacids amino acid linear peptide 42 Asp Tyr Tyr Gly Leu Tyr Tyr Val HisGlu 1 5 10 60 base pairs nucleic acid double linear DNA (genomic) 43TTGTCATGGG ATCTGAATTC CGGGGGGATC GACTATTATG GTTTATATTA TGTTCATGAA 60 20amino acids amino acid linear peptide 44 Leu Ser Trp Asp Leu Asn Ser GlyGly Ile Asp Tyr Tyr Gly Leu Tyr 1 5 10 15 Tyr Val His Glu 20

What is claimed is:
 1. An isolated antibody that binds to an amino acidsequence present in SEQ ID NO:15.
 2. The antibody of claim 1, whereinthe antibody detects or identifies the E2 protein of human papillomavirus 16 (HPV16).
 3. A composition comprising the antibody of claim 1and an antibody binding buffer.
 4. The composition of claim 3, which isa diagnostic composition.
 5. The composition of claim 4, wherein thecomposition is used for identifying, diagnosing, or detecting HPV 16infection.
 6. A monoclonal antibody that binds to an amino acid sequencepresent in SEQ ID NO:15.
 7. The antibody of claim 6, wherein theantibody detects or identifies the E2 protein of human papilloma virus16 (HPV16).
 8. A composition comprising the antibody of claim 6 and anantibody binding buffer.
 9. The composition of claim 8, which is adiagnostic composition.
 10. The composition of claim 8, wherein thecomposition is used for identifying, diagnosing, or detecting HPV 16infection.
 11. A method of identifying, diagnosing, or detecting HPV 16infection, said method comprising contacting a sample from a humansuspected of being infected with HPV 16 with an isolated antibody thatbinds to an amino acid sequence present in SEQ ID NO:15, and detectingbinding of said antibody to said amino acid sequence, wherein bindingindicates an HPV 16 infection.
 12. The method of claim 11, wherein saiddetecting is by enzyme linked immunosorbent assay (ELISA).
 13. Themethod of claim 11, wherein said method detects binding of said antibodywith the E2 protein of HPV
 16. 14. A method of identifying, diagnosing,or detecting HPV 16 infection, said method comprising contacting asample from a human suspected of being infected with HPV 16 with amonoclonal antibody that binds to an amino acid sequence present in SEQID NO:15, and detecting binding of said antibody to said amino acidsequence, wherein binding indicates an HPV 16 infection.
 15. The methodof claim 14, wherein said detecting is by enzyme linked immunosorbentassay (ELISA).
 16. The method of claim 14, wherein said method detectsbinding of said antibody with the E2 protein of HPV 16.